Despite the advent of prophylaxis and HAART therapy, the mortality rate associated with Pneumocystis pneumonia in HIV/AIDS remains similar to that reported prior to the initiation of these therapies. From the close association wit HIV/AIDS, we know that CD4+ T cells are the central effector cell required for immunity against Pneumocystis. Yet the protective CD4+ T cell phenotype and the responses it induces remain elusive, despite 30 years of research. Our recent published work has shown that mice deficient in myeloid Src tyrosine kinases (Src TKO mice) clear Pneumocystis murina more efficiently than control mice as a result of an enhanced alternative macrophage activation (M2a) signature. Our studies now show that M2a polarization develops rapidly after P. murina exposure, yet CD4+ T cells are required for maintaining M2a polarization in the lungs. We further demonstrate a correlation between M2a induction and eosinophil recruitment to the lungs. Higher Epx (eosinophil peroxidase) mRNA expression was observed in Src TKO mice, which have a higher M2a signature and clear P. murina more efficiently. However, Epx was significantly lower in CD4- depleted mice, which have a lower M2a signature and impaired P. murina lung clearance. These data imply a host defense role for M2a-mediated eosinophil recruitment during P. murina lung infection. In turn, we demonstrate in a pilot study that mice lacking eosinophils (dbl GATA1 deficient mice) have impaired P. murina lung clearance. Moreover, we provide new human data showing that CCL22 levels are higher in induced sputum from HIV+ patients colonized with Pneumocystis, which also correlated with higher eosinophil numbers. Finally, we demonstrate strong induction of a true chitinase, acidic mammalian chitinase (AMCase; Chia), in the lungs after P. murina infection. Moreover, induction of AMCase/Chia was elevated in mice with better P. murina clearance and reduced in mice with impaired clearance of P. murina, suggesting an anti- fungal role for AMCase/Chia. Therefore, the goal of this R21 Exploratory/Developmental Research Grant is to define the roles of two M2a host defense components, eosinophil recruitment and AMCase/Chia induction, during the lung immune response P. murina. Our working hypothesis is that early CD4+ T cell responses maintain alternative activation of alveolar macrophages leading to the recruitment of eosinophils and successful elimination of Pneumocystis from the lungs. To test this hypothesis, we propose: (1) to determine the protective capacity of eosinophils during Pneumocystis lung infection and (2) to establish that acidic mammalian chitinase contributes to host defense during Pneumocystis lung infection. PUBLIC HEALTH RELEVANCE: Our data has discovered a new mechanism by which alveolar macrophages are activated in response to Pneumocystis (termed M2a), one of the most significant causes of pneumonia in HIV/AIDS. An effect of the M2a response is the recruitment of eosinophils, which may be an unappreciated effector cell in the host responses to Pneumocystis. Studies in this proposal will determine the specific roles of eosinophils in Pneumocystis lung infection and immune reconstitution associated inflammation.